Understanding the function of mammalian genes requires methods for controlling their expression in the animal. Knockout mice provide important information about many genes, but the lack of graded control and irreversibility of the genetic alteration limits their usefulness, particularly for genes that play important roles in development. This proposal describes a method for creating transgenic mice in which any desired gene can be inhibited in a tissue-specific, reversible, and quantitative manner, using RNA Padlock inhibitors driven by tetracycline (tet)-regulated promoters. RNA Padlocks inactivate the expression of specific genes by forming a topological linkage between an antisense RNA and the target mRNA. In systems where they have been studied, RNA Padlocks have been demonstrated to be much more effective at inhibiting the expression of specific genes than conventional antisense approaches. Proof of principle will be provided by creating a Padlock directed against subunit l of the NMDA receptor (NR-l), which is implicated in learning and neuronal plasticity. A vector expressing this inhibitor under tet control will then be constructed and tested for its ability to inhibit NR-l expression in a neuronal cell line. Demonstration of tet-regulatable inhibition will be followed by the creation of transgenic mice in which NR-l is inhibited specifically in the hippocampus upon administration of a tetracycline derivative. PROPOSED COMMERCIAL APPLICATIONS: Knockout mice are currently the best tool for understanding gene function, yet for many genes knockouts are lethal or prevent normal development. There is thus a need for a method for making conditional knockouts, whereby the gene can be turned off and on at desired times and in specific tissues. Such a method would be invaluable for research purposes and very useful for target validation in drug development. The ability to introduce potent, regulatable gene inhibitors by gene transfer methods is also key to some types of gene therapy, where the aim is to control of a natural gene as needed, at times of a disease flare-up, thus minimizing adverse effects.